Device for detecting different fluorescence signals of a sample support illuminated with different excitation wavelengths

ABSTRACT

Light sources are provided for generating rays of light of different excitation wavelengths, which can be directed toward the sample support by excitation optics. The fluorescent light emitted each time by the sample support can be directed toward a receiver, which generates corresponding fluorescence signals. A mirror assembly with reflective areas and transparent areas is connected between the different light sources and the sample support. The mirror assembly can be displaced in such a manner that the ray of light of a light source passes through a transparent area and reaches the sample support.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/DE01/02982 which has an Internationalfiling date of Aug. 3, 2001, which designated the United States ofAmerica and which claims priority on German Patent Application number DE100 38 185.5 filed Aug. 4, 2000, the entire contents of which are herebyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention generally relates to a device for detectingdifferent fluorescence signals of a sample carrier illuminated bydifferent excitation wavelengths.

BACKGROUND OF THE INVENTION

Wherever quantitative fluorescence immunoassays; for example, arecarried out, sample carriers are known that have a multiplicity ofelectrodes, for example 10,000 electrodes, to which an electric voltagecan be applied selectively. If different sample liquids are led over theelectrodes, different samples can be produced by deposition at theelectrodes, depending on the application of specific voltages. Sincethese samples are marked by two or more fluorescence carriers, theyluminesce differently in the case of excitation by different opticalwavelengths. Biochemical properties can be measured in this way.

It is known in this connection to use dichroic, permanently installedmirrors in order to achieve a separation of the different fluorescencewavelengths that are emitted by the sample carrier. In this case, aproblem exists in that dichroic mirrors can be operated typically onlywhen the beam path is parallel to the position of the dichroic mirrors.In addition, such mirrors are not 100% efficient. At the same time, theyalso require the excitation sources to be electrically clocked.

Sample carriers produced using semiconductor technology are, forexample, built up in several layers and have a multiplicity ofcylindrical platinum electrodes to which it is possible to apply theabovementioned voltages. The sample carriers are arranged in plasticcontainers covered in each case with a glass layer, it being possiblefor the sample liquids to flow through the space between the glass layerand plastic container and come into contact in the process withelectrodes.

Document DE 39 26 090 C2 discloses a dual-beam photometer in which arotatable mirror system divided into silvered and transmitting sectorsis used to split a light bundle issuing from a light source into ameasuring beam and into a reference beam. The two beam paths arerecombined by the same mirror system, the measuring beam penetrating themirror system and passing through a sample to be examined, and thereference beam being reflected at the mirror system and therefore notimpinging on the sample. The recombined beam is detected by a detectordevice. Consequently, the influence of fluctuations in the light sourcebrightness or the detector sensitivity can be eliminated given suitableevaluation of the detected measuring signals. DE 39 26 090 C2 furtherdiscloses in accordance with an exemplary embodiment a dual-beamphotometer having a second light source emitting a continuous spectrum(see FIG. 4), whose radiation is used in a fashion alternating with thefirst light source both as measuring beam and as a reference beam when amirror system divided into four sectors (two silvered and twotransparent sectors) is used. It is possible in this way additionally toachieve compensation of background radiation.

SUMMARY OF THE INVENTION

An embodiment of the present invention includes creating a device fordetecting different fluorescence signals of a sample carrier illuminatedby different excitation waves, in the case of which it may not benecessary to clock the light sources generating the different excitationwavelengths.

In order to detect fluorescence signals of a sample carrier illuminatedby different, preferably two, excitation wavelengths, dichroic mirrorsare not used. In the case of an embodiment according to the presentinvention, a separation of the fluorescence signals is performed withthe aid of a rotatable mirror arrangement that is arranged in the beampath of the excitation and detection optical system and is partlytransmitting in accordance with the number of the excitationwavelengths. This mirror arrangement is moved in the beam path such thatin each case one excitation detection channel is opened and the otherexcitation detection channels are closed. The mirror arrangement to maybe a rotatable mirror.

Further scope of applicability of the present invention will becomeapparent from the detailed description given hereinafter. However, itshould be understood that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description given hereinbelow and the accompanying drawingswhich are given by way of illustration only, and thus are not limitativeof the present invention, and wherein:

FIG. 1 illustrates a schematic of a first side view of a detection partof a device according to an embodiment of the present invention fordetecting two different fluorescence signals of a sample carrierilluminated by two different excitation waves;

FIG. 2 illustrates a side view of the entire device of FIG. 1 at aviewing angle rotated by 90°; and

FIG. 3 illustrates a plan view of a rotatable mirror that is suitablefor a device for detecting two different fluorescence signals in thecase of two different excitation waves.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with FIGS. 1 to 3, an embodiment of the present invention,for detecting two different fluorescence signals in the case of twoexcitation wavelengths, essentially has a first light source 1, a secondlight source 3, and a first excitation optical system 5 that directs thefirst light beam 7 from the first light source 1 onto a sample carrier9. Moreover, the embodiment includes a second excitation optical system11 that directs the second light beam 13 from the second light source 3onto the sample carrier 9, a first filter 15 assigned to a firstfluorescent light, a second filter 17 assigned to a second fluorescentlight, as mirror arrangement a segmented rotatable mirror 19 that inaccordance with FIG. 3 has a first transmitting region 19″ and a secondreflecting region 19′, a first fixed mirror 21 that reflects the firstfluorescent light 23 to the receiver 25, and a second fixed mirror 27that leads the second fluorescent light to the rotatable mirror 19 fromwhich it is reflected to the receiver 25.

The first light source 1 and the second light source 3 may be lasersources, the first light source 1 generating, for example, a laser lightof wavelength 532 nm, and the second light source 3 generating, forexample, a laser light of wavelength 632 nm. The filters 15 and 17 arepreferable steep-edge filters that either allow only the first or thesecond fluorescent light to pass. The first excitation optical system 5comprises a first stop 30 and a first lens arrangement 31 that generatefrom the first laser beam generated by the first light source 1 a firstparallel beam 7, and a first deflecting mirror 33 that directs theparallel beam 7 onto the sample carrier 9 in such a way that the latteris illuminated over its entire surface.

Correspondingly, the second excitation optical system 11 includes asecond stop 34 and a second lens arrangement 35 that generate a secondparallel beam 13 from the second laser beam from the second light source3, and a second fixed deflecting mirror 37 that directs the parallelbeam 13 onto the sample carrier 9 in order to illuminate the entiresurface of the latter.

The rotatable mirror 19 can be rotated about an axis 20 of rotation andhas the reflecting region 19′ and the transmitting region 19″ that, inthe case of the use of two light sources 1, 3 of two differentwavelengths, preferably correspond in each case to half the surface ofthe circular rotatable mirror 19.

The detection optical system 42 includes an optical imaging arrangement43 that is arranged downstream of the sample carrier 9 and generates aparallel beam in each case from the first and second fluorescent light23 and 45, respectively, output by the sample carrier 9, and an opticalimaging arrangement 48 that is arranged upstream of the receiver 25 andprojects the said parallel beams onto the entire surface of the receiver25. The filter 15 and the fixed mirror 27 as well as the filter 17 andthe fixed mirror 21 are part of the detection optical system 42.

The receiver 25 may be a CCD arrangement that, in accordance with thenumber of samples of the sample carrier 9, has photosensitive elementsthat respectively generate a first or second electric fluorescencesignal in accordance with their illumination by the first or secondfluorescent light 23 and 45, respectively. These fluorescence signalsare led to an electronic evaluation system (not illustrated in moredetail).

For example, the sample carrier 9 and the receiver 25 have samples oroptical sensor elements in mutually corresponding raster configurations,the number of samples or sensor elements being of the order of 10 000.

The function of an embodiment of the present invention for separatingtwo fluorescence signals is explained below in more detail.

It is assumed in this case that the reflecting region 19′ is located ina phase in FIG. 1 to the left of the axis 20 of rotation, and thetransmitting region 19″ is located to the right of the axis 20 ofrotation. The result of this is that the first laser beam 7 generated bythe first light source 1 passes through the transmitting region 19″ andis led to the sample carrier 9 by the imaging optical system 5 (FIG. 2)in order to illuminate the entire surface of the latter. The firstfluorescent light 23 emitted by the sample carrier 9 as a consequence ofthe wavelength of the first laser beam 7 is reflected at the reflectingregion 19′ and directed onto the filter 17, passes through the latter,is reflected at the fixed mirror 21, passes through the transmittingregion 19″ of the mirror 19 (FIG. 1) and is directed by the imagingoptical system 48 onto the receiver 25, which generates correspondingfluorescence signals at its individual photosensitive sensor elements.During this phase, the laser beam 13 emitted by the second light source3 is reflected at the reflecting region 19′ such that it cannot reachthe second deflecting mirror 37 and cannot reach the sample carrier 9(FIG. 2).

In the other phase, in which the reflecting region 19′ is located to theright of the axis 20 of rotation, and the transmitting region 19″ islocated to the left of the axis 20 of rotation, the second laser beam 13from the light source 3 passes through the transmitting region 19″ andis directed by the second deflecting mirror 37 onto the sample carrier 9(FIG. 2 with interchanged regions 19′, 19″). The second fluorescentlight 45 generated in this case passes through the transmitting region19″, passes the filter 15, is reflected by the fixed mirror 27 to thereflecting region 19′ of the rotatable mirror 19 and is reflected at thelatter and directed to the imaging optical system 44 (FIG. 1, dottedlines). The latter projects the second fluorescent light 45 onto thereceiver 25. The individual optical sensor elements of the receiver 25then generate corresponding second fluorescence signals. In this phase,the first laser beam generated by the first light source 1 is reflectedat the reflecting region 19′ such that it cannot reach the firstdeflecting mirror 33 and also cannot reach the sample carrier 9.

It is possible in this way to use the rotary movement of the rotatablemirror 19 to switch back and forth between the two laser beams 7 and 13,which are generated simultaneously, in order respectively to be able toilluminate the entire surface of the sample carrier 9, such that in eachcase only one laser beam illuminates the sample carrier 9 and afluorescent light is generated that is led to the receiver 25, while therespective other laser beam is reflect at the reflecting region 19″ ofthe rotatable mirror 19 such that it cannot reach the receiver 25.Consequently, the different fluorescence signals are received insuccessive sequence at the receiver 25 and, if the receiver 25 is a CCDarrangement, are latched to an electronic evaluation device.

It may be pointed out that in order to separate more than twofluorescence signals it is also possible for the rotatable mirror 19 tohave a plurality of transparent and reflecting regions so as to ensurethat in different phases it is always only one fluorescent light that isexcited by a laser beam and led to the receiver, while the respectiveother laser beams are reflected at the reflecting regions such that theycannot excite fluorescent light.

It is also possible to use other movable mirror arrangement instead ofthe rotatable mirror 19 explained. For example, a transparent andreflecting regions can be moved back and forth next to one another in aplate having row, this being done in the direction of the row.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

1. A device for detecting different fluorescence signals of a samplecarrier illuminated over a surface thereof by different excitationwavelengths, the excitation wavelengths having a plurality of samplesexcited by different optical wavelengths to output different fluorescentlight, comprising: a first light source and a second light source beingprovided for generating light beams of different optical wavelengths,the light beams being directed via an excitation optical system toward asurface of a sample carrier; fluorescent light emitted by the samplecarrier being directed to a receiver, wherein said receiver generatesfluorescence signals based on the fluorescent light being projected ontoa photosensitive element of the receiver; and a mirror arrangement withsilvered and transmitting regions being connected between the lightsources and the sample carrier; wherein in a first operating state, afirst light beam of the first light source passes through a transmittingregion to the sample carrier, and a second light beam from the secondlight source is reflected from the silvered region such that it does notreach the sample carrier, and a first fluorescent light is emitted fromthe sample carrier to the reflecting region, which reflects it to afirst fixed mirror, and that the first fixed mirror reflects the firstfluorescent light through the transmitting region to the receiver, thereceiver being screened from the light sources in such a way that thereceiver detects fluorescent light emitted by the plurality of samples,and in a second operating state, the second light beam of the secondlight source passes through the transmitting region to the samplecarrier, and the first light beam from the first light source isreflected from the silvered region such that it does not reach thesample carrier, and a second fluorescent light is emitted from thesample carrier to the reflecting region, which reflects it to a secondfixed mirror, and the second fixed mirror reflects the secondfluorescent light through the transmitting region to the receiver, thereceiver being screened from the light sources in such way that thereceiver detects fluorescent light emitted by the plurality of samples.2. The device as claimed in claim 1, wherein the mirror arrangement is amirror that is rotatable about an axis.
 3. The device as claimed inclaim 2, wherein the rotatable mirror is of circular design and has asemicircular transmitting region and a semicircular reflecting region.4. The device as claimed in claim 2, wherein the first light beam isdirectable via a first excitation optical system to the sample carrierin order to illuminate the entire surface of the latter.
 5. The deviceas claimed in claim 2, wherein the second light beam is directable by asecond excitation optical system to the sample carrier in order toilluminate the entire surface of the latter.
 6. The device as claimed inclaim 2, wherein a first filter is provided between the sample carrierand the receiver in the beam path of the first fluorescent light, thefirst filter passing only the first fluorescent light.
 7. The device asclaimed in claim 1, wherein the excitation optical system includes thefirst light beam being directable via a first excitation optical systemto the sample carrier in order to illuminate the entire surface of thelatter.
 8. The device as claimed in claim 7, wherein the firstexcitation optical system has a first deflecting mirror that directs thefirst light beam to the sample carrier.
 9. The device as claimed inclaim 7, wherein the second light beam is directable by a secondexcitation optical system to the sample carrier in order to illuminatethe entire surface of the latter.
 10. The device as claimed in claim 7,wherein a first filter is provided between the sample carrier and thereceiver in the beam path of the first fluorescent light, the firstfilter passing only the first fluorescent light.
 11. The device asclaimed in claim 1, wherein the excitation optical system includes thesecond light being being directable by a second excitation opticalsystem to the sample carrier in order to illuminate the entire surfaceof the latter.
 12. The device as claimed in claim 11, wherein the secondexcitation optical system has a second deflecting mirror that directsthe second light beam to the sample carrier.
 13. The device as claimedin claim 11, wherein a first filter is provided between the samplecarrier and the receiver in the beam path of the first fluorescentlight, the first filter passing only the first fluorescent light. 14.The device as claimed in claim 1, wherein a first filter is providedbetween the sample carrier and the receiver in the beam path of thefirst fluorescent light, the first filter passing only the firstfluorescent light.
 15. The device as claimed in claim 14, wherein thefirst filter is a steep-edge filter.
 16. The device as claimed in claim1, wherein a second filter is provided in the beam path of the secondfluorescent light between the sample carrier and the receiver, thesecond filter passing only the second fluorescent light.
 17. The deviceas claimed in claim 16, wherein the second filter is a steep-edgefilter.
 18. The device as claimed in claim 1, wherein the receiver is aCCD arrangement including optical sensor elements that are arranged inaccordance with a specific arrangement of the samples of the samplecarrier.